Abstract
Researchers
have shown that the mutations
of TP53 gene is the
most
frequent event in many human cancers and is
associated with a poor clinical
outcome in lung cancer patients. Several studies have revealed
that the distinct
TP53 mutational
pattern between population
groups may be due
to
different racial or exogenous factors.
In this report we studied the mutational spectrum of TP53 gene in 50 lung cancer patients of
a uniquely preserved ethnic population of Kashmir Valley. Germline
TP53 mutational
analyses were also performed to determine the inherited cancer
predisposition. Exons 5-8 of TP53 gene were analyzed
by sequencing DNA of cancerous
tissue and peripheral
blood leukocyte samples
from 50 nonsmall cell lung cancer patients.
The results showed
that TP53 germline mutation
was not found in any patient, indicating
that TP53 germline mutations
were not responsible
for cancer predisposition in this group of patients.
A total of 19 somatic
mutations were found .
No smoking specific characteristic hot spot
codons
were observed except in two
patients. The data thus suggests that TP53
mutations in this study group are induced
by exposure to substances
besides tobacco smoke.
Pesticide exposure may instead be related to tumorigenesis of lung cancer
via TP53 mutations.
Introduction
High incidence
and poor prognosis
of lung cancer
make it a major health
problem worldwide 1,2.
Lung cancer, which was initially
considered an epidemic
disease among men in industrialised nations,
has now become
the leading killer
cancer in both sexes in the United
States and an increasingly common
disease of both sexes in developing countries
3. In Kashmir
valley, the northernmost
part of India,
it ranks second
among all cases in males.
Non-small cell lung cancer accounts
for nearly 85% and small cell lung cancer accounts
for 15% to 20% of cases 4.
The multistage process
of tumorigenesis generally
includes the gain of protooncogenes activity
and the loss or inactivity
of tumor suppressor
genes 5. Among tumor suppressors, TP53 gene abnormalities are the most frequent genetic
events illustrated to date 6-7. TP53 mutation
occurs in about 40-70% of lung cancers
8. Mutations at codons 157, 158, 248, 249, and 273 are more frequently
detected in lung tumors than mutations at other positions
and are considered
”hotspots” for TP53 mutation
9,10,6,11. TP53 cancer-associated mutation in the highly
conserved DNA-binding domain
may prevent or inhibit p53-mediated
cell cycle arrest,
DNA repair, programmed
cell death, and other protective
responses to cell stress and DNA damage.12-14 The causes of TP53 mutations
include both endogenous
factors which contribute
to the infidelity
of DNA synthesis
and exogenous factors
such as chemical
mutagens and radiation
exposure 9. Various
genotoxic compounds have been shown to selectively
induce alterations of specific base pairs in TP53, that are related
to cancer 15. These alterations
can be explained
by the presence
of regionally distinct
carcinogens in both smoke and air, interacting
with local environmental
cofactors, in the development of lung cancer.
Various studies have been conducted
that have revealed
a distinct TP53 mutation
pattern between population
groups 16-18, suggesting
the involvement of a potentially
distinct mutagenic process
in each population.
The present
study aims to identify potential
genetic risk factors
by determining the TP53
germline and somatic
mutations of lung cancer patients
residing in the Kashmir valley
(INDIA). The mutation
spectra of TP53 might provide important
clues for cancer
risk assessment in molecular epidemiology.
Understanding risk factors
and genetic predisposition for lung cancer
is important to lung cancer
prevention.
Subjects and methods
Study subjects
Tumor specimens
(50 cases) and corresponding normal
tissues were obtained
from NSCLC patients
who were admitted
to the Department
of Cardio-vascular and Thoracic Surgery
in Sheri-Kashmir Institute
of Medical Sciences,
Srinagar (INDIA). 5ml of peripheral blood was collected
from each patient.
This research project
has been approved
by the Research
Ethics Committee, of the Institute. Subjects enrolled
in the study were asked to sign informed consent
forms before having
blood samples taken and being interviewed. A detailed questionnaire
was administered by interviewers to all studied
participants about personal
life, occupation, lifestyle,
health and disease,
cigarette smoking and environmental exposures.
The diagnosis of lung cancer
was performed by histological evaluation
of tumour biopsies
and chest computed
tomography scans.
Genomic DNA from peripheral
blood leukocytes and cancerous lung tissues of each patient
was extracted by inorganic salting
out protocol19 or phenol-chloroform protocol
20.
Mutational
analysis
Four primer
pairs were used to amplify
exons 5, 6, 7 and 8 of the TP53
gene (Table I). PCR was carried
out in an Biorad Thermal
cycler at each primer pair’s
respective annealing temperature. PCR-SSCP was performed as follows: initial
denaturation at 950C for 5 min, amplification for 35 cycles
with denaturation at 950C
for
40s, primer annealing
at 550C for exon 5, 620C for exon 6 and 7 and 580C for exon 8 for 70s and elongation
at 720C for 90s in each cycle. The final elongation
was performed at 720C
for 7 min. PCR products after denaturation were analyzed on 8% polyacrylamide
gel (180V) for 5h and stained with AgNO3. Purified
PCR products showing
a mobility shift (20 samples)
in SSCP analysis ( Fig. I) as well as randomly chosen
samples were used for direct DNA sequencing (a total of 45 samples) using the ABI prism 310 automated
DNA sequencer. To confirm mutations reverse sequencing was
also done.
Statistical
analysis
Univariate logistic
regression analysis was used to examine the association between
TP53 mutations
and potential risk factors.
Odds ratio (OR) and their 95% confidence
intervals (95% CI) were estimated.
Statistical analyses were performed using SPSS statistical software (version
11.5).
Results
The characteristics and variable factors
of 50 NSCLC patients from the questionnaire
are summarized in Table
II. A
total of 19 somatic mutations (13
different types) were found in 19 lung cancer patients
(38%). TP53 germline mutations
were not found in extracted
DNA from peripheral
blood leucocytes of any of the 50 patients. The most frequent
type of mutation
was missense (17 cases) and 2 frameshift
mutations. The details of
mutations found are given in Table III. No characteristic hot spot codon were observed except
175 and 282. The TP53
mutation pattern consisted
of 21.05% of A > G transitions
at codon 193, 10.5%
of G > A transitions at codon 175,
A> C transversions at codon 164 each,
15.8% of
G> C transversions
at codon 159 and 221 and C> T
transitions at codon 278, 282 and 294 each and 5.26% of C> G
transv ersions at codon 241, T> C transitions at codon 163 and G> T
transversions at codon 294
each. Also 10.5% of insertion
mutations at codon 260 (insertion
of A) and codon 296 ( insertion
of T) were identified (Fig. II
and III).
All characteristics of the patients
(Table II), were tested for association between
potential risk factors
and TP53 mutations in a univariate
analysis (Table IV). The potential
risk factors associated
with TP53 mutations at the P
